Epitope mapping of the monoclonal antibody FAC2 on the apoprotein of CPa-1 in photosystem II

Using a combination of cyanogen bromide cleavage and endoproteinase digestion we have shown that the putative epitope for the monoclonal antibody FAC2 lies in the region 360Pro-391Ser on the apoprotein of CPa-1. This region lies entirely within the large extrinsic loop of this protein. We have shown previously that the epitope of FAC2 becomes exposed in oxygen-evolving membranes upon treatment with alkaline Tris which releases all four of the manganese associated with the oxygen-evolving site of photosystem II. The epitope is not exposed, however, after CaCl2 treatment and exposure to low concentrations of chloride, conditions which lead to the release of two of the four manganeses associated with the oxygen-evolving site [(1987) Arch. Biochem. Biophys. 256, 295-301]. These results suggest that, upon release of the chloride-insensitive manganese from photosystem II membranes, a conformational change occurs which leads to the exposure of 360Pro-391Ser on CPa-1 to the monoclonal antibody FAC2. © 1989

The psbo1 mutant of Arabidopsis cannot efficiently use calcium in support of oxygen evolution by photosystem II

The Arabidopsis thaliana mutant psbo1 contains a point mutation in the psbO-1 gene (At5g66570) leading to the loss of expression of the PsbO-1 protein and overexpression of the PsbO-2 protein (Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2002) FEBS Lett. 523, 138-142). Previous characterization of fluorescence induction and decay kinetics by our laboratory documented defects on both the oxidizing and reducing sides of Photosystem II. Additionally, anomalous flash oxygen yield patterns indicated that the mutant contains a defective oxygen-evolving complex that appears to exhibit anomalously long-lived S2 and S3 oxidation states (Liu, H., Frankel, L. K., and Bricker, T. M. (2007) Biochemistry 46, 7607-7613). In this study, we have documented that the S2 and S3 states in psbo1 thylakoids decay very slowly. The total flash oxygen yield of the psbo1 mutant was also significantly reduced, as was its stability. Incubation of psbo1 thylakoids at high NaCl concentrations did not increase the rate of S2 and S3 state decay. The oxygen-evolving complexes of the mutant did, however, exhibit somewhat enhanced stability following this treatment. Incubation with CaCl2 had a significantly more dramatic effect. Under this condition, both the S2 and S 3 states of the mutant decayed at nearly the same rate as the wild type, and the total oxygen yield and its stability following CaCl2 treatment were indistinguishable from that of the wild type. These results strongly suggest that the principal defect in the psbo1 mutant is an inability to effectively utilize the calcium associated with Photosystem II. We hypothesize that the PsbO-2 protein cannot effectively sequester calcium at the oxygen-evolving site. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

Carboxylate groups on the manganese-stabilizing protein are required for efficient binding of the 24 kDa extrinsic protein to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein on the binding of the 24 kDa extrinsic protein to Photosystem II were investigated. Carboxylate groups on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The manganese-stabilizing protein which was modified while associated with NaCl-washed membranes could bind to calcium chloride-washed PS II membranes and reconstitute oxygen evolution in a manner similar to that observed for unmodified manganese-stabilizing protein (Frankel, L,K, Cruz, J. C. and Bricker, T. M. (1999) Biochemistry 38, 14271-14278), However, PS II membranes reconstituted with this modified protein were defective in their ability to bind the extrinsic 24 kDa protein of Photosystem II. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified protein and analysis by MALDI mass spectrometry. These studies indicated that the domains 1E-71D, 97D-144D, and 180D-187E are labeled when the manganesestabilizing protein is bound to NaCl-washed Photosystem II membranes. We hypothesize that modified carboxylates, possibly residues 1E, 32E, 139E, and/or 187E, in these domains are responsible for the altered binding affinity of the 24 kDa protein observed

Functional complementation of the Arabidopsis thaliana psbo1 mutant phenotype with an N-terminally His\u3csub\u3e6\u3c/sub\u3e-tagged PsbO-1 protein in photosystem II

The Arabidopsis thaliana mutant psbo1 has recently been described and characterized. Loss of expression of the PsbO-1 protein leads to a variety of functional perturbations including elevated levels of the PsbO-2 protein and defects on both the oxidizing- and reducing-sides of Photosystem II. In this communication, two plant lines were produced using the psbo1 mutant as transgenic host, which contained an N-terminally histidine6-tagged PsbO-1 protein. This protein was expressed and correctly targeted into the thylakoid lumen. Immunological analysis indicated that different levels of expression of the modified PsbO-1 protein were obtained in different transgenic plant lines and that the level of expression in each line was stable over several generations. Examination of the Photosystem II closure kinetics demonstrated that the defective double reduction of QB and the delayed exchange of QBH2 with the plastoquinone pool which were observed during the characterization of the psbo1 mutant were effectively restored to wild-type levels by the His6-tagged PsbO-1 protein. Flash fluorescence induction and decay were also examined. Our results indicated that high expression of the modified PsbO-1 was required to increase the ratio of PS IIα/PS IIβ reaction centers to wild-type levels. Fluorescence decay kinetics in the absence of DCMU indicated that the expression of the His6-tagged PsbO-1 protein restored efficient electron transfer to QB, while in the presence of DCMU, charge recombination between QA- and the S2 state of the oxygen-evolving complex occurred at near wild-type rates. Our results indicate that high expression of the His6-tagged PsbO-1 protein efficiently complements nearly all of the photochemical defects observed in the psbo1 mutant. Additionally, this study establishes a platform on which the in vivo consequences of site-directed mutagenesis of the PsbO-1 protein can be examined. © 2009 Elsevier B.V. All rights reserved

The PsbP domain protein 1 functions in the assembly of lumenal domains in photosystem I

Photosystem I (PS I) is a multisubunit membrane protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase. The PsbP domain protein 1 (PPD1; At4g15510) is located in the thylakoid lumen of plant chloroplasts and is essential for photoautotrophy, functioning as a PS I assembly factor. In this work, RNAi was used to suppress PPD1 expression, yielding mutants displaying a range of phenotypes with respect to PS I accumulation and function. These PPD1 RNAi mutants showed a loss of assembled PS I that was correlated with loss of the PPD1 protein. In the most severely affected PPD1 RNAi lines, the accumulated PS I complexes exhibited defects in electron transfer from plastocyanin to the oxidized reaction center P 700+. The defects in PS I assembly in the PPD1 RNAi mutants also had secondary effects with respect to the association of light-harvesting antenna complexes to PS I. Because of the imbalance in photosystem function in the PPD1 RNAi mutants, light-harvesting complex II associated with and acted as an antenna for the PS I complexes. These results provide new evidence for the role of PPD1 in PS I biogenesis, particularly as a factor essential for proper assembly of the lumenal portion of the complex. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc

High yield non-detergent isolation of photosystem I-light-harvesting chlorophyll II membranes from spinach thylakoids: Implications for the organization of the PS I antennae in higher plants

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Styrene-maleic acid copolymer was used to effect a non-detergent partial solubilization of thylakoids from spinach. A high density membrane fraction, which was not solubilized by the copolymer, was isolated and was highly enriched in the Photosystem (PS) I-light-harvesting chlorophyll (LHC) II supercomplex and depleted of PS II, the cytochrome b6/f complex, and ATP synthase. The LHC II associated with the supercomplex appeared to be energetically coupled to PS I based on 77 K fluorescence, P700 photooxidation, and PS I electron transport light saturation experiments. The chlorophyll (Chl) a/b ratio of the PS I-LHC II membranes was 3.2 ± 0.9, indicating that on average, three LHC II trimers may associate with each PS I. The implication of these findings within the context of higher plant PS I antenna organization is discussed

Natively oxidized amino acid residues in the spinach PS I-LHC I supercomplex

© 2020, Springer Nature B.V. Reactive oxygen species (ROS) production is an unavoidable byproduct of electron transport under aerobic conditions. Photosystem II (PS II), the cytochrome b6/f complex and Photosystem I (PS I) are all demonstrated sources of ROS. It has been proposed that PS I produces substantial levels of a variety of ROS including O2.−, 1O2, H2O2 and, possibly, •OH; however, the site(s) of ROS production within PS I has been the subject of significant debate. We hypothesize that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in spinach PS I which was isolated from field-grown spinach. The modified residues were identified by high-resolution tandem mass spectrometry. As expected, many of the modified residues lie on the surface of the complex. However, a well-defined group of oxidized residues, both buried and surface-exposed, lead from the chl a’ of P700 to the surface of PS I. These residues (PsaB: 609F, 611E, 617M, 619W, 620L, and PsaF: 139L, 142A,143D) may identify a preferred route for ROS, probably 1O2, to egress the complex from the vicinity of P700. Additionally, two buried residues located in close proximity to A1B (PsaB:712H and 714S) were modified, which appears consistent with A1B being a source of O2.−. Surprisingly, no oxidatively modified residues were identified in close proximity to the 4Fe–FS clusters FX, FA or FB. These cofactors had been identified as principal targets for ROS damage in the photosystem. Finally, a large number of residues located in the hydrophobic cores of Lhca1–Lhca4 are oxidatively modified. These appear to be the result of 1O2 production by the distal antennae for the photosystem